Chemical Compositionand Anti-acetyl cholinesterase Activity of Flower Essential Oils of Artemisiaannuaat Different Flowering Stage.

The chemical composition of the essential oils of flower at the pre-flowering, full-flowering and post-flowering stage of A. annua was analyzed by GC and GC/MS and sixty-two components were identified. The main compounds in the pre-flowering oil were β-myrcene (37.71%), 1, 8-cineole (16.11%) and camphor (14.97%). The full-flowering oil contained predominantly caryophyllene (19.4%), germacrene D (18.1%), camphor (15.84%), 1, 8-cineole (10.6%) and (Z)-β-farnesene (9.43%). The major constituents identified in the post-flowering oil were camphor (16.62%), caryophyllene (16.27%), β-caryophyllene oxide (15.84%), β-farnesene (9.05%) and (-)-spathulenol (7.21%). The variety of anti-AChE activity of flower oil of A. annua at three flowering stage might be a result of the variety of the content and interaction of those terpenoids with anti-AChE activity. The greatest acetylcholinesterase inhibitory activity (IC50 = 0.13 ± 0.02 mg mL(-1)) was exhibited by the essential oil of flower of A. annua at post-flowering stage.

Alzheimer is a progressive degenerative neurologic disorder resulting in impaired memory and behavior. Epidemiological data indicate a potentially considerable increase in the prevalence of the disease over the next two decades (5). Most treatment strategies have been based on the cholinergic hypothesis which postulated that memory impairments in patients of cholinergic function in brain. One of the most promising approaches for treating this disease is to enhance the acetylcholine level in the brain by means of using acetylcholinesterase (AChE) inhibitors (6). Several AChE inhibitors are being investigated for the treatment of Alzheimer. However, only tacrine, donepezil, rivastigmine, and galanthamine have been approved by the Food and Drug Administration in the United States (7). These compounds have been reported to have adverse effects including gastrointestinal disturbances and problems associated with bioavailability (8), which reinforces the interest resources.
In this study, our focus was on evaluating acetylcholinesterase inhibitory properties of A. annua phases, as there are no reports on AChE inhibitory activity of A. annua. We also determined the chemical composition of the essential oils by capillary gas chromatography coupled to mass spectrometry (GC-MS).

Plant materials
A. annua cultivar (Wuling-3938) used in this study, grew in the Artemisia annua GAP Cultivation Demonstration Site of Holleypharm, capitula organs, leaves and stem of A. annua, and China and deposited in the Herbarium, College of Bioengineering, Chongqing University, Chongqing, China.

Main instrument and reagent
AChE, tacrine and sodium lauryl sulfate MO). Acetylthiocholine iodide (ATCI) was [2-nitrobenzoic acid] (DTNB) was obtained from ACROS. All organic solvents (analyticalreagent grade) were purchased from Chongqing -1 phosphate buffer (PB) with pH of 7.4 was used as a buffer throughout the experiment unless otherwise stated. AChE used in the assay was -1 protein). The lyophilized enzyme was prepared -1 stock solution. The enzyme stock solution was kept at -20 º C. The further enzyme-dilution was dissolved in -1 BSA in buffer. ATCI and DTNB were -1 -1 stock solutions, respectively.

Essential oil extraction
The essential oils from three samples were obtained by hydrodistillation during 6 h using a Clevenger-type apparatus (9). The yield of each essential oil was determined on average over the three replicates. These oils were dried over analysis.

Microplate assay for AChE activity
The AChE inhibitory activity of esstial oils was screened by Ellman's colorimetric method -1 oscillation mixing, 37 º C pre-incubation 10 min, -1 substrate, 37 º C SDS to terminate reaction. The absorbance was measured with microplate reader at 405 nm when the reaction reached the equilibrium. A control reaction was carried out using water instead of extract. The obtained absorbance value was considered 100% activity. Inhibition (%) was calculated in the following way: I% = (100 -(Asample/Acontrol)) × 100 Where Asample is the absorbance of the reaction containing the extract and Acontrol is l the absorbance of the reaction control. Tests were carried out in triplicate and a blank with phosphate buffer (PB) instead of enzyme solution was done. Extract concentration providing 50% inhibition (IC 50 ) was obtained plotting the inhibition percentage against extract solution concentrations.
Analysis of the essential oils GC analysations were performed using a Shimadzu GC-2010 gas chromatograph equipped with an FID and an HP-5 fused silica column with a 5% phenyl-substituted methylpolysiloxane phase. The oven temperature was programmed The carrier gas, helium (99.999%), was adjusted to a linear velocity of 43 cm/sec. The essential diluted solution was injected into the GC/MS in the split mode with a split ratio of 1/20. MS analyses were performed using a Shimadzu MS-QP2010 with ionization energy of 70 eV, a scan time of 0.5 s and a mass range of 33-450 amu (Atomic mass unit/Dalton (u/Da)).
comparison of their mass spectra with those of the spectrometer database using the NIST147 mass spectral database and also with those of authentic through comparing the fragmentation patterns and Retention index with those reported in the literature (12-14). The percentages of compounds were calculated by the area normalization method without considering response factors to establish abundances. The retention index was found with a standard mixture of C8 to C22 compounds under chromatography conditions, consistent with those of the chromatography conditions of the analyzed samples. For each essential oil, the RI and the peak area percentages were calculated as mean values of the three injections.

Results and Discussion
Extraction yields Essential oils obtained by the conventional stages of A. annua. with 2.21%, 1.42% and 1.25% yield (w/w), respectively.

Chemical composition of the essential oils
In this work, the chemical composition of the three essential oil samples from A. annua was analyzed by GC-MS. Thirty-six, forty-two representing 98.88%, 99.27% and 96.57% of the phases, respectively. Table 1    Acetylcholinesterase inhibitory activity Acetylcholine is a compound liberated at the synaptic gap as a neurotransmitter.
pathological features in central nervous system disorders. The most important changes observed in the brain are a decrease in cortical levels of the neurotransmitter acetylcholine. Therefore, The AChE inhibitory activity of the essential oils A. annua has never been reported in the past. Essential oil of this plant was tested to determine their ability as acetylcholinesterase inhibitors and the results are depicted in Table 2. The greatest inhibitory activity was exhibited by 50 = 0.13 ± 0.02 -1 ). Analysis of the results shows that these oils are moderate AChE inhibitors. Galantamine, a compound used pharmacologically, showed an IC 50 In previous reports, it has been mentioned that 1,8-cineole, camphor, -pinene, -pinene, borneol, linalool, bornyl acetate, linalyl acetate, menthone, carvone, anetole, anisole, eugenol, nonyl alcohol, isomenthol, (-)-menthol, (+)-menthol, citronellol, -myrcene, terpinene, 3-carene, -caryophyllene and -caryophyllene oxide have anti-AChE activity (17)(18)(19)(20). It was reported that 1, 8-cineole / -pinene and 1, 8-cineole/caryophyllene oxide combinations were minor synergy. In contrast, a combination of camphor and 1, 8-cineole was antagonistic. This study shows that the high concentration of 1, 8-cineole and the low concentration of camphor in the oil may result in an increase in its anticholinesterase activity (17)  ) a , of essential oils.
. a : Averages ± SD were obtained from three different experiments